Electrode for dielectrophoretic apparatus, dielectrophoretic apparatus, method for manufacturing the same, and method for separating substances using the electrode or dielectrophoretic apparatus

ABSTRACT

To provide an electrode for a dielectrophoretic apparatus in which a background detected by reflecting an excited light on an electrode present under the substance (molecule) is reduced and an S/N ratio is enhanced. Also, there is provided an dielectrophoretic apparatus, in an apparatus in which a liquid containing substances to be separated is present in a non-uniform electric field formed by a dielectrophoretic electrode, and separation is carried out by a dielectrophoretic force exerting on the substances, wherein the collecting ability of substances is enhanced. The present invention is characterized in that a vacant space is provided in an electrode whereby substances subjected to influence by a negative dielectrophoretic force can be concentrated in said vacant space of an electrode, or above or below portion of the space.

CROSS REFERENCE TO RELATED APPLICATION

This application is a Divisional Application of prior application Ser.No. 11/064,828, filed on Feb. 25, 2005, which is a DivisionalApplication of prior application Ser. No. 09/833,566, filed on Apr. 13,2001, now U.S. Pat. No. 6,875,329, issued on Apr. 5, 2005, which isbased upon and claims priority of Japanese Applications Nos. 2000-112337filed on Apr. 13, 2000, and No. 2000-374210 filed Dec. 8, 2000.

BACKGROUND OF THE INVENTION

This invention relates to an electrode for a dielectophoretic apparatus,in which a background can be reduced to enhance an S/N (Signal/Noise)ratio in detecting a substance to be measured (molecules to be measured)by a fluorescent strength or the like, a method for manufacturing thesame, an electrode constitution provided with the electrode, and amethod for separating substances using the electrode.

This invention further relates to an dielectrophoretic apparatus havingan enhanced collecting ability, a method for manufacturing the same, anda method for separating substances using the apparatus.

Processing technology of materials at scales of nanometer to micrometerby means of micromachining technology such as photolithography hasrecently been established by development of semiconductor technologiesand it has still continued its progress at present.

In the fields of chemistry and biochemistry, new technology called aMicro Total Analysis System (μ-TAS), Laboratory on a chip is growing, inwhich such micromachining technology is employed to carry out a wholeseries of chemical/biochemical analytical steps of extraction ofcomponents) to be analyzed from biological samples (extraction step),analysis of the component(s) with chemical/biochemical reaction(s)(analysis step), and subsequent separation (separation step) anddetection (detection step) using a highly small analytical deviceintegrated on a chip having each side of a few centimeters to a few tencentimeters in length.

Procedures of the μ-TAS are expected to make a large contribution tosaving the analyzing time, reducing the amounts of samples to be usedand reagents for chemical/biochemical reactions, and reducing the sizeof analytical instruments and the space for analysis in the course ofall the chemical/biochemical analytical steps.

For the separation step in μ-TAS, in particular, there have beendeveloped capillary electrophoretic methods in which a capillary (finetube) with an inner diameter of less than 1 mm which is made of Teflon,silica, or the like as material is used as the separating column toachieve separation with charge differences of substances under a highelectric field, and capillary column chromatographic methods in which asimilar capillary is used to achieve separation utilizing the differenceof the interaction between carrier in the column medium and substances.

However, capillary electrophoretic methods need a high voltage forseparation and have a problem of a low sensitivity of detection due to alimited capillary volume in the detection area and also these is foundsuch a problem that they are not suitable for separation of highmolecular weight substances, though suitable for separation of lowmolecular weight substances, since the length of capillary forseparation is limited on the capillary column on a chip and thus acapillary can not be made into a length enough for separating highmolecular weight substances. In addition, in capillary columnchromatographic methods there is a limit in making the throughput ofseparation processing higher and also there is such a problem thatreducing the processing time is difficult.

Thus, attention has recently been paid to a method for solving theproblems as described above, which comprises utilizing such a phenomenonso-called dielectrophoretic force that a positive and negativepolarization occurs in substances placed under a non-uniform electricfield, thereby providing a driving force of moving the substances [H. A.Pohl, “Dielectrophoresis”, Cambridge Univ. Press (1978); T. B. Jones,“Electromechanics of Particles”, Cambridge Univ. Press (1995), and thelike].

These separation methods are presently believed to be the suitableseparation method in μ-TAS from the following points: (1) a rapidseparation can be expected at a low applied voltage without requiring ahigh voltage as in capillary electrophoresis, since an electric fieldand its gradient can be increased to an extreme extent if micromachinedelectrodes are employed, because the degree of dielectrophoretic forcesdepends on the size and dielectric properties of substances (particles)and is proportional to the electric field gradient; (2) an increase intemperature due to applying the electric field can be minimized, since astrong electric field area is localized at a significantly small region,and a high electric field can be formed; (3) as the dielectrophoreticforce is a force proportional to the electric field gradient, the forceis understood as independent on the polarity of the applied voltage, andthus works under an AC electric field in a similar way to a D.C.electric field, and therefore if a high frequency A.C is employed, anelectrode reaction (electrolytic reaction) in an aqueous solution can besuppressed, so that the electrodes themselves can be integrated in thechannel (sample flow path); (4) improvement in a detection sensitivitycan be expected, since there is no restriction to a chamber volume ofthe detection component unlike the capillary electrophoresis, and thelike.

The dielectrophoresis termed herein is a phenomenon in which neutralparticles move within non-uniform electric field, and the force exertingon molecules is called a dielectrophoretic force. The dielectrophoreticforce is divided into two forces, i.e., a positive dielectrophoreticforce in which substances move toward a high electric field, and anegative dielectrophoretic force in which substances move toward a lowelectric field.

(General Equation of Dielectrophoretic Forces)

The equivalent dipole moment method is a procedure of analyzingdielectrophoretic forces by substituting induced charges for anequivalent electric dipole. According to this method, thedielectrophoretic force F_(d) acted upon a spherical particle with aradius of a which is placed in an electric field E is given by:

F _(d)=2πa ³ε_(m) Re[K*(ω)]∇(E ²)   (1)

wherein K*(ω) means by using an angular frequency of the applied voltageω and the imaginary unit j as follows:

K*(ω)=ε_(p)*−ε_(m)*/ε_(p)*+2Ε_(m)*   (2)

ε_(p) *=ε _(p) −jσ _(p)/ω, ε_(m)*=ε_(m) −jσ _(m)/ω  (3)

wherein ε_(p), ε_(m), σ_(p), and σ_(m) are permittivity and conductivityof the particle and the solution, and complex quantities are designatedby *.

Equation (1) indicates that in a case of Re [K*(ω)]>0, the force worksin such a way as attracting the particle toward a strong electric fieldside (positive dielectrophoretic, positive DEP), and in a case ofRe[K*(ω)]<0, the force works in such a way as pushing the particletoward a weak electric field side (negative dielectrophoretic, negativeDEP).

As will be apparent from the above-described Equations, whether thepositive electrophoresis occurs in a certain substance or the negativeelectrophoresis occurs therein is decided by the interaction of threeparameters, i.e., 1) frequency of an electric field applied, 2)conductivity and permittivity (dielectric constant) of medium, and 3)conductivity and permittivity (dielectric constant) of substance.

When these parameters are changed, even the same substance shows apositive dielectrophoresis or a negative dielectrophoresis. The negativedielectrophoresis is a phenomenon in which the substance moves toward alow electric field which is weak in density of electric flux line whilethe positive dielectrophoresis moves toward a high electric field whichis high in density of electric flux line. FIG. 1 is a view forexplaining the negative dielectrophoresis. The negativedielectrophoretic force is a force for carrying substances to such afield as to be lowered where the density of electric flux line receivedby the substance.

Sometimes, the substances are measured by concentrating them in an areawhere an electric field on an electrode is weak by using the negativedielectrophoresis as described and thereafter measuring them byfluorescent strength or the like. The detection of the fluorescentstrength is carried out by irradiating an excitation light on thesubstance to be measured to observe fluorescent light from the uppersurface of the electrode.

At that time, where a conventional electrode is used, there poses aproblem that the excitation light is reflected even on the electrodewhich is present under the substance to be measured, and thus reflectedlight is detected as a great background. This leads to a problem ofreducing the measurement sensitivity. Besides, where a conventionalelectrode is used, since light does not permeate through the electrode,the substances concentrated (gathered) on the electrode cannot bedetected by absorbance.

Further, the dielectrophoresis is contemplated to be a separation methodsuitable for μ-TAS. However, In consideration of a case of applicationof the dielectrophoresis to μ-TAS, it is extremely important to enhancethe collecting ability. In this respect, the conventionaldielectrophoretic apparatus should not yet be satisfied.

That is, if the collecting ability of substances is enhanced, separationbecomes enabled in the electrode region, and the substances are heldefficiently, whereby separation with high S/N (Signal/Noise) ratio isrealized. Further, for example, particularly, in the Field-Flowfractionation for carrying out separation by the interaction of thedielectrophoretic force and the fluid drag exerting on the substances,separation in a short electrode region can be made even at the same flowvelocity.

SUMMARY OF THE INVENTION [Invention 1]

It is an object of the present invention to provide an electrode for adielectrophoretic apparatus which reduces a background in which anexcitation light is reflected on an electrode which is present under asubstance (a molecule) and detected to enhance an S/N ratio.

It is a further object of the present invention to provide an electrodefor a dielectrophoretic apparatus, which can be detected even byabsorbance.

It is another object of the present invention to provide a method forseparating substances and a detection method using the above electrode.

For achieving the aforementioned objects, the present inventors havestudied earnestly, as a result of which the inventors have thought outthat an electrode in an area where substances to be measured areconcentrated (gathered) is removed to thereby enable reduction inbackground caused by reflection of an excitation light from theelectrode.

In the past, there are many patents and articles in connection withapparatus and method in a dielectrophoretic chromatography apparatus(Field-Flow fractionation), but a dielectrophoretic apparatus and methodwhich reduces a background by removing an electrode including an areawhere substances to be measured are concentrated to enhance an S/N ratioare not known at all, and such an idea is not known at all.

The present invention is characterized in that by forming a vacant spacein an electrode, substances subjected to influence by a negativedielectrophoretic force generated by application of voltage to theelectrode are concentrated in the vacant space of the electrode, orabove or below position of the space.

The vacant space is formed from a hollow space or formed of a materialwhich does not substantially reflect excitation light or permeates lightto such an extent as capable of measuring the absorbance. However, thevacant space is preferably a hollow space.

The space where substances subjected to influence by the negativedielectrophoretic force are concentrated is a space in which the densityof electric flux line is low for the substances.

Further, through all the substances subjected to influence by thenegative dielectrophoretic force are preferably concentrated in thevacant space, concentrated substances in the vacant space may be a partof all the substances.

The electrode constitution of the present invention is characterized bycomprising an electrode, and a lid provided thereabove so as to form agap between the lid and said electrode surface, the electrode beingformed as in the electrode of the present invention provided with thevacant space.

The electrode constitution of the present invention includes anelectrode of the present invention, a substrate (an electrode baseplate) and a lid. In the dielectrophoretic apparatus, a device forapplying a voltage to an electrode and a detection section are added tothe electrode or the electrode constitution.

A method for manufacturing an electrode according to the presentinvention characterized in that said vacant space is formed by physicalor chemical means.

The separation method and detection method according to the presentinvention are characterized in that using the electrode of the presentinvention provided with the vacant space, a liquid including substancessubjected to influence by the negative dielectrophoretic force generatedby application of voltage to the electrode is positioned in theelectrode or the vacant space or in the vicinity thereof, or causes toflow thereabove or therebelow, whereby substances subjected to influenceby the negative dielectrophoretic force are concentrated (gathered) inthe vacant space, or above or below position of the space.

The separation method of the present invention can be used for liquidsin which two kinds or more of substances are dissolved or suspended, butpreferably, the substances subjected to influence by the negativedielectrophoresis force concentrated in the vacant space or in avertical direction thereof are granular substances. Because, in thegranular substances, an area in which the density of electric flux lineis low and the granular substances are concentrated tends to be thevacant space or in a vertical direction thereof.

The vacant space of the present invention, should be formed in such away that an area in which the density of a electric flux line is low andthe granular substances are concentrated may be formed in the vacantspace or in a vertical direction thereof by changing the size of thesubstances subjected to influence by the negative dielectrophoresisforce, and the width and depth of an electrode used (the height from theelectrode surface to the lid part and or the height from the vesselbottom to the electrode surface) and frequently applied.

However, particularly, where the substances to be measured aredissolved, for example, in liquid such as water, preferably, thesubstances subjected to influence by the negative dielectrophoresisforce are bound to the substances to be measured in a sample through“substances binding to the substances to be measured” to form a complex,and a reaction substance including the complex is applied to thedielectrophoresis.

It is noted that the substances to be measured used in the presentinvention means substances (molecules) to be concentrated in the area inwhich the density of electric flux line is low, and need not always bean object for measurement.

[Invention 2]

It is a further object of the present invention to provide, in anapparatus for enhancing the collecting ability of substances in which aliquid containing substances to be separated is present within anon-uniform electric field formed by a dielectrophoretic electrode toseparate the substances by the dielectrophoretic force exerting on thesubstrate.

For achieving the aforementioned objects, the present inventors havestudied earnestly, as a result of which the inventors have thought outthat a base plate (substrate) of among electrodes are excavated to forma part lower than the electrode level whereby the non-uniform electricfield region is increased and the drag of fluid is reduced to enhancethe collecting ability.

In the past, there are many patents and articles in connection withseparation apparatus and method making use of a dielectrophoretic force,particularly, apparatus and method in Field-Flow fractionation, but anapparatus and method which enhances the collecting ability by forming “alower level place than an electrode level” are not known at all, andsuch an idea is not known at all.

Preferably, the present invention provides a dielectrophoretic apparatushaving an electrode provided on a substrate, wherein means for realizingan increase of an non-uniform electric field region is formed among theelectrodes.

The means for realizing an increase of a non-uniform electric fieldregion is characterized in that a lower level places than the electrodelevel is formed among the electrodes. The “lower level place than theelectrode level” is formed whereby electric fields are formed not onlyabove between the electrodes but below thus increasing a non-uniformelectric field region, and further, where for example, Field Flowfractionation is used, since the flow velocity of fluid in that placesdrops, the fluid drag is reduced to enhance the collecting ability ofsubstances.

For forming “lower level places than electrodes level”, a base plate(substrate) may be excavated between electrodes by physical and/orchemical means to form the lower level place than the electrode levelamong the electrodes. The physical means termed herein is, for example,a method for excavation using a suitable knife or the like, for example,an LIGA (Lithographile Galvanoformung Abformung) method usingsynchrotron radiant light. Further, the chemical means is etching forexcavating a base plate using an etching liquid for a base plate.Further, for example a base plate can be excavated by etching usingplasma of a reaction gas [Reactive ion etching (RIE)] formed by a highfrequency power supply, in which a physical excavation and chemicalexcavation are conducted at the same time. It is noted that the means asdescribed above may be suitably combined to carry out excavation of abase plate.

Further, a separation method according to the present invention is aseparation method for substances in which a liquid containing substancesto be separated is present within a non-uniform electric field formed bythe dielectrophoretic electrode, and separation is carried out due to adifference in a dielectrophoretic force exerting on the substancescharacterized in that an increase of a non-uniform electric field regionis realized by lower level places than electrode level formed between(or among) electrodes, to thereby enhance the collecting ability.

Dielectrophoresis (DEP) termed herein is a phenomenon in which a neutralparticle moves within a non-uniform electric field by interaction ofconductivity and dielectric constant of substances, conductivity anddielectric constant of media, and frequency applied, and a force actingon the particle is called a dielectropherotic force. Thedielectrophoretic force is divided into two kinds, i.e., a positivedielectrophoretic force in which substances move toward a high electricfield, and a negative dielectrophoretic force in which substances movetoward a low electric field.

In the following, a case where a positive dielectrophoretic force exertson a molecule will be described.

Namely, as shown in FIG. 2, a neutral molecule placed in an electricfield has a positively induced polarization charge +q downstream in theelectric field and a negatively induced polarization charge −q upstreamin the electric field, respectively, thus +q receives a force of +qEfrom the electric field E and this portion is pulled upstream in theelectric field. If the molecule is neutral, +q and −q have an equalabsolute value, and if the electric field is uniform regardless of thepositions, both received forces are balanced, therefore the moleculedoes not move. However, in the case where the electric field isnon-uniform, an attractive force toward a strong electric field becomeslarger, thus the molecule is driven toward the strong side of theelectric field.

As described above, the molecule in a solution variously moves within anelectric field according to the dielectrophoretic force generated in themolecule. However, for example, in the Field-Flow fractionation, themovement of molecules is governed by three factors: thedielectrophoretic force F_(d), the force F_(v) generated by the drag dueto the flow in the flow path, and the force F_(th) due to the thermalmovement. {circle around (1)} in the case of F_(d)>>F_(v)+F_(th),molecules are captured (trapped) on the electrode, {circle around (2)}in the case of F_(d)<<F_(v)+F_(th), molecules are eluted out with flowin the flow path, regardless of the electric field. {circle around (3)}in the case of F_(d)≈F_(v)+F_(th), molecules are carried downwards withrepeating adsorption and desorption on the electrode, so that themolecules arrive at the outlet with delay, relative to the set flow inthe flow path.

In the present invention, since a portion between electrodes isexcavated deeply whereby a non-uniform electric field is formed belowbetween the electrodes, the non-uniform electric field region isincreased and the flow of fluid in that portion becomes slow to reducethe drag force Fv of fluid, whereby Fd becomes further great under thecondition {circle around (1)} as described above and Fv becomes furthersmall thus enhancing the collecting rate. Further, the particles trappedin the electric field formed below between electrodes are hard to flowout since the particles are positioned at “lower level places thanelectrode level”.

The above and other objects and advantages of the invention will becomemore apparent from the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an explanatory view of the negative dielectrophoresis.

FIG. 2 is a view showing the principle of the positivedielectrophoresis.

FIG. 3 is a plan view showing an embodiment of an electrode of thepresent invention.

FIG. 4 is a plan view showing a further embodiment of an electrode ofthe present invention.

FIG. 5 is a plan view showing another embodiment of an electrode of thepresent invention.

FIGS. 6A and 6B are plan views showing examples of a conventionalelectrode.

FIGS. 7A and 7B are plan views showing further examples of aconventional electrode.

FIGS. 8A and 8B are plan views showing other examples of a conventionalelectrode.

FIGS. 9A and 9B are plan views showing still other examples of aconventional electrode.

FIGS. 10A and 10B are plan views showing other examples of aconventional electrode.

FIGS. 11A through 11G are plan views showing still other examples of aconventional electrode.

FIG. 12 is an explanatory view in the case where fluorescent measurementis made according to the method of the present invention, (A) showingthe case where a fluorescent measuring unit is provided above, (B)showing the case where a fluorescent measuring unit is provided below.

FIG. 13 is a plan view showing an electrode of the present inventionprepared in Example 1.

FIG. 14 are respectively, a plan view (A) and a sectional view (B)showing a further embodiment of the present invention.

FIG. 15 is a sectional view showing an example of “lower level placesthan electrode level” of the present invention formed by isotropicetching (A), anisotropic etching (B), and RIE or LIGA (C),

FIGS. 16A through 16E are plan views showing electrodes used in thepresent invention.

FIG. 17 is a sectional view of a dielectrophoretic chromatographyapparatus.

FIG. 18 is a sectional view showing an example of forming “lower levelplace than electrode level” on a base plate (substrate) according to themethod of the present invention.

FIG. 19 is a graph showing a relationship between etching time and thedepth of a groove measured in Example 3.

FIG. 20 is a graph which measured the collecting rate with respect tobovine-serum albumin (BSA) protein, using the dielectrophoreticchromatography apparatus according to the present invention and theconventional dielectrophoretic chromatography apparatus.

FIG. 21 is a graph which measured the collecting rate with respect to500 bp DNA, using the dielectrophoretic chromatography apparatusaccording to the present invention and the conventionaldielectrophoretic chromatography apparatus.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The preferred embodiments of the present invention will be describedhereinafter.

First, the invention 1 will be described in detail hereinafter.

FIG. 3 is a plan view showing an embodiment of an electrode for thedielectrophoretic apparatus of the present invention, showing an examplein which a hollow space (a vacant space) 12 is formed in a part 13 onwhich are concentrated substances (substances to be measured) subjectedto influence by the negative dielectrophoretic force generated by anelectrode 11 having many hexagonal portions associated.

The hollow space 12 is formed so as to form an area which is low indensity of electric flux line in which the substances to be measured maybe concentrated in the hollow space 12 or in a vertical directionthereof. The area which is low in density of electric flux line is anarea which is lower in density of electric flux line than that of anelectrode in the circumference, and in general, an area which is lowestin density of electric flux line. The size of the hollow space 12 isdifferent depending on the kind and size of substances to be measured,the distance between an electrode base plate and a cover glass (depth)or the like, but is generally formed to be larger than a space 13 onwhich are concentrated the substances to be measured when the hollowspace is not formed. The hollow space 12 may be communicated as shown inFIG. 3 or may be independent every hexagonal portion as shown in FIG. 4.

In the hollow space 12, all the circumference may be surrounded by theelectrode or a break 14 may be present in a part as shown in FIG. 3, butpreferably, all the circumference may be surrounded by the electrode.

When all the circumference of the vacant space is surrounded by theelectrode, electric flux lines are generated from the circumference ofthe vacant space, and therefore, the vacant space is to be surrounded bya high electric field region so that the substances tend to beconcentrated on a specific portion and may be collected easily.

On the other hand, where a space of the vacant space is not surroundedby the electrode, no line of electric force is generated from thatportion, and therefore, a portion which is not a high electric fieldregion is generated, and the substances may be easily moved through thatportion. Therefore, there is a case where the intended substance is hardto be collected.

As the size of substances (particles, molecules) to be concentrated onthe hollow space is small, attention should be paid to the width of anelectrode. Because an area above the electrode will be a portion whichis low in density of electric flux line for the substance than thehollow space. The reason why is that since a electric flux line is alsogenerated from an edge of an electrode in contact with the hollow space,a degree of influence caused by the electric flux line generated from anedge of an electrode in contact with the hollow space is differentdepending on the size of the substance. Where the substances to beconcentrated on the hollow space are small, this problem can be solvedby narrowing the width of an electrode having the hollow space.

The shape of the electrode and the hollow space may be a circle, oval ora polygon, the shape of which is not particularly restricted. Also, thewidth of the electrode itself may be wider or a thin like a wire. Inshort, the construction of an electrode may be employed so that anelectrode is not present in an area in which detected objects subjectedto the negative dielectrophoretic force are concentrated, and in avertical direction thereof.

Since even the same electrode construction, there appears a differencein a region where the measured objects are concentrated due to thechange of the frequency of the electric field applied, and conductivityand dielectric constant of the measured object and the medium, theelectrode construction may be decided according to the frequency of theelectric field applied according to the using object. Converselyspeaking, the substances to be measured can be concentrated at thedesired position by varying the frequency or the like adjusting to theelectrode construction.

Preferably, the hollow space 12 may be formed in the electrode, forexample, by physical means such as a cutting method using, for example,a suitable knife or the like and embossing method, chemical means suchas etching for removing an electrode, for example, using an etchingliquid, or for example, by physical and chemical means such as ReactiveIon Etching (RIE) using a reactive gas formed into plasma by a highfrequency power supply, and so on.

The electrode formed with the vacant space 12 of the present inventionis preferably prepared, for example, by the fine processing technique(Biochim. Bophys. Acta. 964,231-230 and so on) as described below:

-   (A) For example, a resist is coated on a base plate having copper,    gold, aluminum or the like laminated thereon, and an electrode    photomask is laminated on the resist. Then, light is irradiated to    expose and develop the resist to dissolve a resist corresponding to    a vacant space and a portion other than the electrode, which is then    dipped into an etching liquid to apply etching to the electrode    surface (aluminum surface), and the remaining resist on the    electrode surface is removed. It is noted that the resist may be a    positive resist for removing a portion exposed to light or a    negative resist for removing a portion not exposed.-   (B) Lift off method    -   After a resist is coated on a base plate, an electrode photomask        is laminated on the resist, to which is applied exposure. Then        development is carried out to remove a resist corresponding to        an electrode portion, and an electrode material is laminated on        the whole upper surface by vapor deposition or sputtering. Then,        a resist corresponding to a portion other than the electrode and        a vacant space (an electrode is laminated on the upper surface)        is removed.-   (C) Metal mask method    -   A metal mask with only the electrode portion applied with        hollowing is laminated on a base plate, on which upper surface        is coated with an electrode material by vapor deposition or        sputtering. Then, the metal mask (an electrode material is        laminated on the upper surface) is removed.

In the present invention, an electrode is one made of conductivematerials such as, for example, aluminum, gold, copper and the like. Itsstructure can be any structure capable of causing dielectrophoreticforces, that is, forming a horizontally and vertically non-uniformelectric field, including, for example, an interdigital shape [J. Phys.D: Appl. Phys. 258, 81-89 (1992); Biochim. Biophys. Acta., 964, 221-230(1988), and the like].

The electrode of the present invention is, preferably, formed on theupper surface and/or the lower surface of the base plate (substrate).Normally, since the liquid containing the substance to be measured iscaused to flow above the electrode, an electrode formed on the uppersurface of the base plate is used. However, an electrode is placed in astate that floated in hollow, and the liquid containing the substance tobe measured can be flown below the electrode. In this case, an electrodeformed on the lower surface of a base plate or on both upper and lowersurface of a base plate is used.

The electrodes used in the present invention include, for example, anelectrode in the shape having many electrodes of the same shape(hexagon) associated, as shown in FIGS. 3 and 4, and an electrode formedsuch that a cathode and an anode are provided internally and externally,respectively, and longitudinal and lateral parts are made to the same orsomewhat different, as shown in FIG. 5.

Since in the electrode as shown in FIGS. 3 and 4, negativedielectrophoretic regions can be formed in not only one place butseveral places, several hollow spaces having an area which is low indensity of the same electric flux line can be prepared, whereby thefluorescent strength of several places is measured and averaged tothereby obtain data with reliability.

Further, in an electrode provided with a cathode and an anode internallyand externally, respectively, as shown in FIG. 5, there is one measuringplace, but since a space require is small, that can be contributed tointegration of measurement of many inspected objects.

Other concrete examples of electrodes as shown in FIGS. 3 and 4 includea shape in which many triangular outwardly projecting parts areassociated in a spaced relation opposite to upper and lower portion of alinear web as shown in FIG. 6, a shape in which many trapezoidaloutwardly projecting parts are associated in a spaced relation oppositeto upper and lower portion of a linear web as shown in FIG. 7, a shapein which many hexagons are associated linearly as shown in FIG. 8, ashape in which many square outwardly projecting parts are associated ina spaced relation opposite to upper and lower portion of a linear web asshown in FIG. 9, and a shape in which many semicircular outwardlyprojecting parts are associated in a spaced relation opposite to upperand lower portion of a linear web as shown in FIG. 10. While in (A) and(B) in FIGS. 6 to 10, shapes of ends are different, but either of themwill suffice.

Further, other concrete examples of electrodes as shown in FIG. 5include, for example, as shown in FIGS. 11(A) to (G), electrodes inwhich an external anode is formed to be polygon such as square andoctagon, circle, semi-circle, and oval; and as an internal cathode, acathode head located in a central part of the cathode is formed to bepolygon such as square and octagon, circle and the like. In the presentinvention, any electrode can be used as long as the elect-rode itselfcan be used for dielectrophoresis for forming a hollow space, and thekind of electrodes is not restricted.

A base plate (substrate) used when an electrode is prepared is notparticularly restricted if it can be used in this field, and a baseplate formed of a non-conductive material, for example, such as glass,plastics, quartz, silicon or the like is preferred.

The base plate may be formed of a transparent material, but a materialneed not always be a transparent material if excitation light is notsubstantially reflected, or light is permeated to such an extent ascapable of measuring absorbance.

The electrode may be similar to prior art except formation of a vacantspace, and an organic layer may be formed on the electrode to preventadsorption of various materials on the electrode.

For manufacturing the electrophoretic apparatus of the present inventionusing the electrode of the present invention formed with the vacantspace as described above, those other than the electrode may be formedin a manner similar to prior art.

For embodying the separation method of the present invention using theelectrode and the dielectrophoretic apparatus of the present inventionformed with the vacant space as described above, the separation methoditself may be carried out in a manner similar to prior art.

Namely, a liquid containing substances to be separated, a liquid inwhich for example, two or more kinds of substances (molecules orparticles) are dissolved or suspended is placed in presence within anon-uniform electric field formed using the electrode as describedabove, and separation may be accomplished due to a difference in thedielectrophoretic force exerting on the substances. It is noted that anelectric field applied in the present invention may be either DCelectric field or AC electric field, but AC electric field is preferred.

In the separation method of the present invention, granular substancesof 100 nm to 100 μm are easily concentrated on an area which is lower indensity of electric flux line. Because the granular substances havingthe size to some extent may easily concentrated on an electrode havingan area which is low in density of electric flux line in whichsubstances to be measured are concentrated in the vacant space and aboveor below position of the space. However, it is possible, even whensubstances to be separated or measured are small particles or molecules,to constitute an electrode capable of forming an area which is low indensity of electric flux line in upper and lower directions of thevacant space by narrowing the width of an electrode or deepening thedepth (the distance between the electrode base plate and the cover glassand/or the distance from the vessel bottom to the electrode). In short,since the influence of electric flux line received by particles isdifferent according to the size of particles, when the particle havingthe size to some extent is applied to the separation method of thepresent invention, an electrode in which the particles are concentratedin the vacant space or in upper and lower directions thereof can beeasily formed.

Accordingly, for separating molecules or small particles, which aremeasured materials, in a solution of molecules or a suspension of smallparticles, a complex in which substances to be measured (through“substances binding to substances to be measured”, if necessary) arebound to substances subjected to influence by the negativedielectrophoretic force, preferably, granular substances having the sizeof 100 nm to 100 μm is subjected to the separation method using adielectrophoresis. This is, because of the fact that if the size ofparticles is too small, the width of the electrode need be extremelynarrowed.

The granular substances are bound as described above whereby thesubstances are enlarged, and so, separation of the substances to bemeasured is facilitated. Accordingly, the granular substances functionas substances for enhancing separation.

The granular substance used in the present invention includes inorganicmetal oxides such as silica and alumina; metals such as gold, titanium,iron, and nickel; inorganic metal oxides and the like having functionalgroups introduced by silane coupling process and the like; living thingssuch as various microorganisms and eukaryotic cells; polysaccharidessuch as agarose, cellulose, insoluble dextran; synthetic macromolecularcompounds such as polystyrene latex, styrene-butadiene copolymer,styrene-methacrylate copolymer, acrolein-ethylene glycol dimethacrylatecopolymer, styrene-styrenesufonate latex, polyacrylamide, polyglycidylmethacrylate, polyacrolein-coated particles, crosslinkedpolyacrylonitrile, acrylic or acrylic ester copolymer,acrylonitrile-butadiene, vinyl chloride-acrylic ester and polyvinylacetate-acrylate; relatively large biological molecules such aserythrocyte, sugars, nucleic acids, proteins and lipids, and the like.

The “granular substance” are normally bound to “substance binding tosubstance to be measured” for use. By doing so, it can be bound to“substance to be measured” in a sample. However, the granular substancemay be bound directly to the substance to be measured by a chemicalbinding method, for example, such as a method for introducing afunctional group into the surface of the granular substance andafterwards binding through the functional group, or a binding method thegranular substance to the substance to be measured through a linker.

Further, for binding the granular substance to the “substance binding tothe substance to be measured”, a method similar to a method for labelingthe measured substance by a labeling substance described later may beemployed.

Where a substance having properties capable of specifically binding tothe substance to be measured directly is used as the granular substance,the operation as described above is unnecessary. The granular materialas described includes, for example, neucleic acid, protein, lipid and soon.

The “substance binding to the substance to be measured” used in thepresent invention is bound to the granular substance for use to form acomplex of the substance to be measured, the “substance binding to thesubstance to be measured”, and the granular substance from the substanceto be measured in a sample, and a complex of a molecule other than thesubstance to be measured, the “substance binding to the substance to bemeasured” and the granular substance may be not formed substantially,which is not particularly restricted. In short, even if being bound tothe substances other than the substance to be measured, it will sufficeif that may not form the aforesaid three complex substance. However, itis actually preferred that the “substance specifically binding to thesubstance to be measured is used.

A “substance binding to the substance to be measured” refers to asubstance binding to the “substance to be measured ” by interactionssuch as an “antigen”-“antibody” reaction, a “sugar chain”-“lectin”reaction, an “enzyme”-“inhibitor” reaction, a “protein”-“peptide chain”reaction, and a “chromosome or nucleotide chain”-“nucleotide chain”reaction. If one partner is the substance to be measured in eachcombination described above, the other is a “substance binding to thesubstance to be measured” as described above.

For forming a complex of binding the substance to be measured in asample with the granular substance directly or through the “substancebinding to the substance to be measured”, a sample containing thesubstance to be measured, the granular substance and, if necessary the“substance binding to the substance to be measured” are, for examplerespectively dissolved, dispersed or suspended in water or a bufferliquid, for example, such as tris (hydroxymethyl amino methane) buffers,a Good's buffer, a phosphate buffer, borate buffer into a liquidmaterial, and these liquid material may be mixed and contacted with eachanother.

The separation method of the present invention is roughly divided intotwo methods as follows:

[Separation Method 1]

First, where the substance to be measured, or the complex of thesubstance subjected to influence of the negative dielectrophoretic force(substance for enhancing separation) and the substance to be measured(through “substance binding to the substance to be measured”, ifnecessary) exhibits the same negative dielectrophoretic force as that ofthe substance other than the substance to be measured, in case of thesubstance to be measured or the complex showing the greaterdielectrophoretic force than that of the substance other than thesubstance to be measured, only substantially the substance to bemeasured, or substance for enhancing separation and the complex ofsubstance for enhancing separation and the substance to be measuredreceive the great dielectrophoretic force and are separated.

Namely, for example, by suitably setting the electric field strength andthe medium conditions in such a way that the substance to be measured orthe complex substance of the substance subjected to influence of thenegative dielectropherotic force and the substance to be measured(through “substance binding to the substance to be measured, ifnecessary) is concentrated in the vacant space above thedielectropherotic electrode or in the upper and lower directionsthereof, but that the substances other than the substance to be measuredare not concentrated, these substance to be measured and the substanceother than the substance to be measured can be separated.

The method of the present invention is suited for separation in thestate free from flow. However, the so-called dielectrophoreticchromatography apparatus (Field Flow Fractionation apparatus) whichcarries out separation by the interaction of the dielectrophoretic forcegenerated in molecules by the electric field and the movement ofmolecules, may be used to carry out separation. In this case, bysuitably setting the flow velocity (speed is made slow) in such a waythat only substance to be measured or the complex of the substancesubjected to influence of the negative dielectrophoretic force and thesubstance to be measured (through “substance binding to the substance tobe measured, if necessary) is collected in the vacant space of theelectrode or in the upper and lower directions by the dielectrophoreticforce, these substance to be measured and the substances other than thesubstance to be measured can be separated. In the condition that thesubstance trapped in the hollow space of the electrode or in the upperand lower directions thereof is not moved by the flow, many samples canbe applied to the hollow space of the electrode by the measurement inthe flow, thus enhancing the measurement sensitivity.

[Separation Method 2]

Second, where the substance to be measured or the complex of thesubstance subjected to influence by the negative dielectropherotic forceand the substance to be measured (through “substance binding to thesubstance to be measured”, if necessary) is one subjected to influenceby the negative dielectropherotic force different from substances otherthan the substance to be measured, namely where the substance to bemeasured or the complex of the substance for enhancing separation(substance subjected, to influence by the negative dielectropheroticforce) and the substance to be measured exhibits the negativedielectropherotic force and the substances other than the substance tobe measured exhibits the positive dielectropherotic force, either of{circle around (1)} the substance to be measured or the complex of thesubstance to be measured and the substance subjected to influence by thenegative dielectropherotic force and {circle around (2)} the substancesother than the substance to be measured moves to the hollow space or inthe upper and lower directions thereof while the other moves to adifferent electrode region whereby the substance to be measured can beseparated from the substances other than the substance to be measured.

When the substance to be measured separated by the separation methodaccording to the present invention can be detected by a method accordingto properties own by the substance, the presence or absence of thesubstance to be measured contained in a sample can be measured(detected).

Namely, using the dielectrode according to the present invention, thedielectrode constitution and the dielectrophororetic apparatus, a liquidmaterial(sample) containing the substance subjected to influence by thenegative dielectropherotic force generated by application of voltage tothe electrode [or substance to be measured or the complex of thesubstance for enhancing separation and substance to measured (through“substance binding to the substance to be measured, if necessary”)] islocated at the electrode according to the present invention, or thevacant space or in the vicinity thereof, or is caused to flow above orbelow thereof, whereby the substances subjected to influence by thenegative dielectrophoretic force are concentrated on the vacant space,above or below thereof, and afterwards, the substance to be measured ina sample can be detected by optically detecting the substance.

The substance to be measured in the above-described method is that canbe measured by any optical method, or that can be labeled by anoptically detectable labeling substance, or bound to the “substancebinding to the substance to be measured” that can be measured(detected), or that can be labeled by an optically detectable labelingsubstance.

In the present invention, the substance to be measured or the “substancebinding to the substance to be measured” may be labeled by the opticallydetectable labeling substance, and labeling itself may be carried out bya well-known labeling method generally carried out in a conventionalmethod generally used in the field of, for example, well-known EIA, RIA,FIA or a hybridization method.

The optically detectable labeling substances which can be used in thepresent invention are any substances usually used in the art of enzymeimmunoassay (EIA), fluoroimmunoassay (FIA), hybridization method, andthe like, and are not particularly limited. However, the labelingsubstance capable of being detected by the fluorescent strength, thelight emission strength or the absorbance is particularly preferred.

In the above-described method, as the “substance binding to thesubstance to be measured”, the “substance binding to the substance to bemeasured” that can be measured (detected) by any optically detectablemethod or that can be labeled by an optically detectable labelingsubstance is generally used.

More concretely, the detection method according to the present inventionmay be carried out in a manner as described below.

The substance to be measured or the complex of the substance to bemeasured and the separation enhancing substance (if necessary, throughthe substance binding to the substance to be measured and/or thesubstance binding to the substance to be measured labeled by theoptically detectable labeling substance) obtained by reacting thesubstance to be measured and the separation enhancing substance (ifnecessary, and the substance binding to the substance to be measuredand/or the substance binding to the measured substance labeled by theoptically detectable labeling substance) and the substances other thanthe substance to be measured (for example, the free substance binding tothe substance to be measured or the free labeled substance to bindingthe substance to be measured) are separated according to the separationmethod of the present invention as mentioned above. Next, the separatedsubstance to be measured or the separated complex is optically detectedon the basis of properties of the substance to be measured or thesubstance binding to the substance to be measured (or the labelingsubstance binding to the substance binding to the substance to bemeasured in the complex) in the complex to measure the presence orabsence of the substance to be measured in the sample.

Further, according to the present invention, not only the presence ofthe substance to be measured in the sample can be detected, but also theamount of the substance to be measured in the sample can be measuredquantitatively. The quantitative measurement of the substance to bemeasured may be done similarly to prior art where the complex is notformed, and in case where the complex substance is formed, the followingmethod may be employed.

That is, the substance to be measured or the complex of the substance tobe measured and the separation enhancing substance (if necessary,through the substance binding to the substance to be measured and/or thelabeled substance binding to the measured substance) and the substancesother than the substance to be measured [for example, the free substancebinding to the substance to be measured (or the free labeled substancebinding to the substance to be measured)] are separated according to theseparation method of the present invention as described above. Next, theamount of the separated substance to be measured or the substancebinding to the substance to be measured in the complex (or the opticallydetectable labeling substance binding to the substance binding to thesubstance to be measured in the complex), or the amount of the freesubstance binding to the substance to be measured (or the opticallydetectable labeling substance binding to the free labeled substancebinding to the substance to be measured) are obtained by the opticalmeasurement method according to these properties, and the amount of thesubstance to be measured in the sample can be obtained on the basis ofthe obtained amount.

In the above-described method, in order to obtain the amount of thesubstance to be measured in the sample on the basis of obtained amountsof the substance to be measured, the substance binding to the substanceto be measured or the labeling substance, for example, the quantity ofspecific molecules in the sample may be calculated, by using acalibration curve showing a relationship between the amount of thesubstance to be measured, and the amount of the substance binding to thesubstance to be measured in the complex (or the labeled substancebinding to the substance to be measured) or the amount of the freesubstance binding to the substance to be measured (or the opticallydetectable labeling substance in the labeled substance binding to thesubstance to be measured), obtained by carrying out the same measuringmethod mentioned above except for using a sample whose concentration ofthe substance to be measured is known.

According to the present invention, the substance to be measured(molecules to be measured) can be concentrated in the hollow space ofthe electrode or in the upper and lower directions thereof. When theexcitation light is irradiated on the concentrated measured molecules,since the electrode is not present under the molecules, the backgroundcaused by being reflected even on the electrode is not detected, ascompared with the case using the conventional electrode, as shown inFIG. 12(A). As a result, the S/N ratio is enhanced, as compared withprior art and the measuring sensitivity is enhanced.

Further, if the electrode of the present invention is used, since theelectrode is not present under the substances to be measured, afluorescent detector can be provided on the opposite side as shown inFIG. 12(B). Further where it is provided on the opposite side, the S/Nratio is enhanced (slit effect) since the parts other than the regionwhere the substances to be measured are concentrated are covered withthe electrode, whereby in said parts the excitation light irradiatedfrom the upper surface does not reach the lower surface, and therefore,the background can be reduced.

Further, according to the present invention, since the measurement canbe done from the lower surface, the absorbance of the substances to bemeasured is measured, which has been heretofore impossible, to enableqaualitative (detection) and quantitative measurement of the substancesto be measured.

In this case, the S/N ratio is further enhanced (slit effect) since theparts other than the region where the substances to be measured areconcentrated are covered with the electrode, whereby in said parts lightdoes not permeate through the electrode from the upper surface to thelower surface, and therefore, the background can be further reduced.

In the following, the invention 2 will be described in detail.

FIG. 14 shows an embodiment of the present invention, showing an examplein which an electrode 3 is supported in a lengthwise spaced relation bya convex member 2 (a support column) on a substrate (a glass substrate)1.

A “lower level place than electrode level” (a communication groove) 4which is semicircular in section is formed between the electrodes 3, 3,as shown in FIG. 14(B), and communication grooves 4, 4 adjacent to eachother are communicated at parts other than the convex member 2, as shownin FIG. 14(A). However, alternatively, the electrode 3 is supported by awall (a convex member) 2′, and grooves 4′, 4′ adjacent to each other areisolated by the wall 2′ so as not to be communicated, as shown in FIG.15(B).

In the embodiments shown in FIGS. 14 and 15, portions other than theconvex members 2 and 2′ are formed on the “lower level place thanelectrode 3 level” (4 and 4′).

However, a concave portion (hole) may be singly or in plural in a spacedrelation provided in a part between the electrodes 3, 3, but preferably,the whole or a major portion between or among electrodes is formed in alower level place than the electrode (4 or 4′) level as shown in FIGS.14 and 15 to enhance the collecting ability.

Where the concave portion (hole) is formed in a part between theelectrodes 3, 3, preferably, it may be formed in a minimum gap 5 betweenthe electrodes. Since this portion is high in electric field strength,if the concave portion (whole) is formed in this portion, the collectingability is further enhanced. However, if that is formed in the wholeincluding this portion, further the collecting ability can be enhanced,because a portion for trapping molecules increases.

The width of the groove 4 (the same as the distance between theelectrodes 3, 3 in the case shown in FIGS. 14 and 15) is suitablydecided according to the size of substances as separated substances bythe dielectrophoresis and is said absolutely though giving great effectto the electric field strength. In the substance of the size which ismicrometer, the width is preferably, 1 time to 100 times of the diameterof the substance, more preferably, 1 time to 10 times. Further, in caseof a bio-molecule such as a protein, a gene or the like, for example,such as a peptide, a protein or the like, normally, the width is 1 nm to10 μm, preferably 1 nm to 5 μm. In case of nucleotide chain(polynucleotide, oligonucleotide), normally, the width is 1 nm to 100μm, preferably 1 nm to 50 μm.

Generally, if the depth is deeper, a portion for trapping a moleculeincreases. Further, particularly, in case of Field-Flow fractionation,the flow velocity at the groove portion is suppressed to enhance thecollecting ability (collecting rate). However, if being too deep, whereit is necessary to measure a molecule trapped on the electrode by thedielectrophororesis, the molecule trapped is sometimes hard to bereleased from the groove portion or not released. Accordingly, the depthof the groove is, preferably, 1/1000 times to 10 times of the width ofthe groove, more preferably, 1/1000 times to 1 time.

With respect to the depth of the groove, if isotropic etching is usedfor formation as shown in FIGS. 14 and 15, when the groove is made morethan the width of the electrode, the convex member which holds theelectrode is totally dug away whereby the electrode 3 is peeled off.Accordingly, when the groove is formed by this method, the depth of thegroove is set to ½ or less of the maximum electrode width.

Where anisotropic etching of a silicon wafer is used for formation, asshown in FIG. 15(B), etching progresses only in a direction of depth atan angle of about 55 degrees. Accordingly, where etching is made by thismethod, the maximum distance depthwise (the distance betweenelectrodes÷2)×1.42 (tan 55 degrees) results.

As shown in FIG. 15(C), where formation is made by RIE or LIGA, etchingprogresses substantially vertically. Accordingly, where etching is madeby these methods, the depth of the groove is in the range describedabove, namely, preferably, 1/1000 times to 10 times, more preferably1/1000 times to 1 time.

The spacing of the groove (=width of the electrode itself) is notaffected by the separated object if limiting to separation by thepositive dielectrophororesis. It is normally from the processingaccuracy in the fine processing technique to 1 nm to 50 μm, morepreferably, 1 nm to 10 μm.

The groove by the isotropic etching shown in FIG. (A) is formed byetching a glass base plate or a plastic base plate. In the isotropicetching, various shapes are formed according to the extent of etchingsuch as the case where the electrode 3 is supported by the wall 2 on thebase plate and the grooves 4, 4 adjacent to each other are formed so asto be isolated by the wall 2, or the case where the electrode 3 issupported by the convex member 2 on the base plate, and the grooves(communication grooves) 4, 4 adjacent to each other are communicated.

The groove by the anisotropic etching shown in FIG. 15(B) is formed byetching a silicon base plate. In this case, the electrode 3 is supportedon the wall 2′ on the base plate, and the grooves 4′, 4′ adjacent toeach other are isolated by the wall 2′.

The groove by RIE shown in FIG. 15(C) is formed by etching a silicon orSi0₂ base plate, and the groove by LIGA is formed by etching polymer,ceramic, plastic base plate etc. In these cases, the electrode 3 issupported on the wall 2″ on the base plate, and the grooves 4″, 4″adjacent to each other are isolated by the wall 2″.

In the isotropic etching shown in FIGS. 14 and 15(A), generally, thegroove or the communication groove 4 is formed to have a shape whosesection is semicircular, or semi-oval. When a groove is formed by theanisotropic etching shown in 15(B), generally, the groove 4′ issubjected to etching into a substantially V-shape finally via asubstantially trapezoid in section. When a groove is formed by RIE orLIGA shown in FIG. 15(C), generally, etching is made to a substantiallysquare in section. Accordingly, various sectional shapes are formedaccording to the way of etching and the way of forming “a lower levelplace than electrode level”, but in the present invention, the shape of“a lower level place than electrode level” (such as a communicationgroove, a groove, a concave part, etc.) are not particularly limited.

A wall or a convex member 2 in FIG. 15(A) is formed into a shape inwhich a central part is bound; a wall 2′ in FIG. 15(B) is formed into atrapezoidal shape; and a wall 2″ in FIG. 15(C) is formed into a squareshape, but the wall, the convex member 2, the wall 2′, and the wall 2″may be any shape as long as they can support the electrode 3, and arenot particularly limited.

The electrode 3 used in the present invention is formed of a conductivematerial, for example, such as aluminum, gold or the like, and theconstruction thereof will suffice to be one which produce thedielectrophoretic force, that is, a non-uniform electric field inhorizontal and vertical directions, for example, an interdigital shape[J. Phys. D: Appl. Phys. 258, 81-88, (1992), Biochim. Biophys. Acta.964, 221-230, (1988), etc.} being listed.

More concretely, preferable are, as shown in FIG. 16, (A) a shape inwhich many triangular outwardly projecting parts 7 a are formed in aspaced relation opposite to upper and lower parts of a linear web-likepart 6; (B) a shape in which many square outwardly projecting parts 7 bare formed in a spaced relation opposite to upper and lower parts of alinear web-like part 6; (C) a shape in which many trapezoidal outwardlyprojecting parts 7 c are formed in a spaced relation opposite to upperand lower parts of a linear web-like part 6; (D) being sine wave shapeat upper and lower portions, a shape in which many sine wave convexparts 8 and concave parts 9 (concave part 9 and convex part 8) areformed linearly opposite to upper and lower portions; and (E) beingsaw-tooth shape at upper and lower portions, a shape in which manyconvex parts 8′ of saw-tooth and concave parts 9′ (concave part 9′ andconvex part 8′) are formed linearly opposite to upper and lowerportions. However, any shape can be used if the electrode can be usedfor dielectrophoresis, and the shapes are not particularly limited.

Such an electrode as described is normally prepared by providing a pairor more electrodes having shapes as described above on comb-tooth-wiseon a base plate formed of a non-conductive material, for example, suchas glass, plastic, quartz, silicon, etc. by using known fine processingtechnique [Bichim. Bioophys. Acta., 964, 221-230, etc.]. Further, thedistance between the electrodes 3 opposite (adjacent) to each other isnot particularly limited as long as a non-uniform AC electric field ofstrong electric field strength can be formed, and should be suitably setaccording to the kind of molecules intended.

The thickness of the electrode 3 may be similar to prior art, andconcretely, the thickness is normally 0.5 nm or more, preferably, 0.5 nmto 1000 nm, more preferably, 1 nm to 1000 nm.

The electrode 3 may be similar to prior art except the thickness, and anorganic layer may be formed on the electrode in order to preventadsorption of various materials on the electrode.

The dielectrophoretic apparatus according to the present invention maybe manufactured in a manner similar to prior art except “a lower levelplace than electrode level” (such as a communication groove 4, a groove4′, a concave portion etc.) such as a flow path and a dielectrophoreticelectrode.

The “lower level place than electrode level” may be formed, for example,by excavating a base plate between electrodes by means of physical meanssuch as an excavating method using a suitable knife or the like, a LIGA(Lithographile Galvanoformung Abformung) method using a synchrotronradiant light and an embossing method using a suitable embossing die ;chemical means for excavating a base plate, for example, using anetching liquid for a base plate; or physical and chemical means such asetching using reactive gases formed into plasma by a high frequencypower supply [Reactive Ion Etching (RIE)].

It is noted that the above-described means may be combined suitably tocarry out excavation of a substrate.

As an etching liquid, a known etching liquid may be selected accordingto material of a substrate. Where a lower level place than electrodelevel is formed in a part of a substrate, etching may be accomplishedwith masking is suitably applied to a portion which is not desired to beexcavated.

For embodying the separation method of the present invention using thedielectrophoretic apparatus according to the present invention, theseparation method itself is the same as prior art.

That is, a liquid containing a substance to be separated, for example, aliquid in which more than two kinds of substances (molecules orparticles) are dissolved or suspended is present in a non-uniformelectric field formed using the electrode (electrode base plate) asdescribed above whereby separation may be accomplished by a differenceof the dielectrophoretic force exerting on the substances.

Generally, a non-uniform electric field is formed horizontally andvertically within a flow path on the substrate to cause to flow a liquidcontaining a substance to be separated from an inlet, and separation maybe accomplished by a difference of the dielectrophoretic force exertingon the substances. However, of course, the substance may be separatedinto a component held in a specific portion of an electrode and acomponent not held for carrying out separation without generating aflow.

For separating by a difference of the dielectrophoretic force exertingon the substances (molecules, particles), the substance may be separatedinto a molecule etc. held in a specific portion of an electrode and amolecule etc. not held. Or, since molecules subjected to a strongerdielectrophoretic force move later than molecules subjected to a weakdielectrophoretic force, separation may be accomplished making use ofthe fact that a difference is produced in moving time.

As shown by an arrow in FIG. 17, when a liquid containing a substance tobe separated in a direction crossing the lengthwise of an electrode iscaused to flow into a flow path of the apparatus according to thepresent invention, the flow velocity in the communication passage(groove) 4 becomes slower than that of the flow path portion so that thedrag Fv of fluid applied to the molecule entered the communicationgroove 4 can be reduced. Further, by the provision of the communicationgroove 4 between the electrodes 3, 3, the range affected by the electricfield becomes widened, and the space where the trapped molecules arestocked becomes widened whereby the collecting rate (ability) isenhanced.

The measuring method of the present invention may be carried out inconformation with the known method as described above other than thatusing the separation method of the present invention, and the reagentsused may be suitably selected from the well-known reagents.

While the present invention will be further described hereinafterconcretely with reference to examples and reference examples, thepresent invention is not at all limited thereto.

EXAMPLES Example 1 Preparation of an Electrode of the Present InventionFormed with a Vacant Space by Etching

The electrode according to the present invention was prepared by coatinga resist on a glass base plate applied with aluminum vapor deposition,then exposing through laminating a photomask having an electrode andvacant space pattern depicted by an electron beam depicting device onthe resist, and developing the resist, dissolving a resist filmcorresponding to the vacant space and portions other than the electrode,and thereafter dipping it into an etching liquid to apply etching to analuminum surface, and removing the resist remained on the aluminumsurface to form an electrode having a vacant space shown in FIG. 13. Thepattern of the vacant space was changed to prepare electrodes 1 to 4different in length (μm) of a) to e) in FIG. 13. Table 1 shows thelength (μm) of a) to e) of electrodes 1 to 4 prepared.

TABLE 1 Electrode 1 Electrode 2 Electrode 3 Electrode 4 (μm) (μm) (μm)(μm) a 14 8 8 8 b 8 2 2 2 c 5 5 10 15 d 2 2 2 2 e 3.5 3.5 3.5 3.5

Example 2 Dielectrophoretic Test of Beads on a Hollow Electrode

Where beads having a diameter of 1 μm was subjected to dielectrophoresisusing a conventional electrode, beads are concentrated (gathered) at aposition on the electrode whose field strength is weak. In the design ofthe electrode prepared in Example 1, the aluminum electrode portion in aregion where the beads are gathered are excluded.

A dielectrophoretic test was conducted under the electric field that thebeads show the negative dielectrophoresis on the electrode (electrode 2in Table 1) prepared in Example 1, using beads having a diameter of 1 μmwith the fluorescent-labeled surface thereof.

A sample solution with the beads suspended was dropped above theelectrode substrate (hollow space), and afterward, a cover glass wasput, and observation was made by an optical microscope.

As a result of observation of the dielectrophoretic test, it has beenconfirmed that the beads were concentrated in the hollow space (vacantspace) of the electrode by the negative dielectrophoretic force. Thebeads were concentrated while floating in the solution above the hollowspace (near the cover glass).

Reference Example 1 Manufacture of Dielectrophoretic Electrode Substrate

A multi-electrode array having a minimum gap of 7 μm, an electrode pitchof 20 μm, and the number of electrodes of 2016 (1008 pairs) wasdesigned, and a photomask according to the design was made formanufacturing the electrode as follows.

On a glass substrate on which aluminum was deposited and to which aphotoresist was applied, an electrode pattern as designed was drawn onan electron beam drawing machine, and then the photoresist was developedand the aluminum was etched to make the photomask.

The electrode substrate was manufactured according to the methoddescribed in T. Hashimoto, “Illustrative Photofabrication”, Sogo-denshiPublication (1985), as follows.

The photomask thus made was contacted tightly with thealuminum-deposited glass substrate to which a photoresist was applied,and then exposed to the electrode pattern with a mercury lamp. Theelectrode substrate was manufactured by developing the exposed glasssubstrate for the electrode and etching the aluminum surface, followedby removing the photoresist remained on the aluminum surface.

Example 3 Formation of “Lower Level Place than Electrode Level” on aSubstrate by Etching

As shown in FIG. 18, etching was applied to the glass substrate 1 of thedielectrophororetic electrode prepared in a manner described inReference Example 1 to form a communication groove 4 in a portion amongthe electrodes 3 on the glass substrate 1.

As an etching liquid, sodium fluoride sulfuric acid (NH₄F 3%, H₂SO₄,H₂0) was used. Sodium fluoride sulfuric acid has properties to dissolveboth glass and aluminum, but since the speed for etching glass is veryquick as compared with that for etching aluminum, a glass portion otherthan the aluminum electrode can be subjected to etching with an aluminumelectrode as a mask.

It is observed that in case where the thickness of aluminum of anelectrode is 40 nm, when etching to the depth of 3 μm or more is done,an electrode is bent by a flow of water when the etching liquid iswashed with pure water. However, in case of thickness of 250 nm, thephenomena that the electrode is bent was not observed.

A relationship between an etching time (sec.) and the depth (μm) of acommunication groove formed between electrodes, upon etching, wasmeasured. The result indicated that the etching time and the depth of agroove to be formed are in a proportional relation as shown in FIG. 19.The depth of a groove was measured by cutting an electrode with a glasscutter and observing its section with a microscope.

Reference Example 2 Manufacturing an Electrode Substrate Having a FlowPath

In order to separate molecules by the movement of the molecules under annon-uniform electric field, a flow path on the electrode substratemanufactured in Example 3 was made using silicone rubber.

The silicone-rubber flow path for sending a solution containingdissolved molecule on the electrode had a depth of 25 μm and a width of400 μm and was designed such that the flow path runs through a region inwhich the electrode on the electrode substrate was placed.

Its manufacturing was carried out according to the method described inT. Hashimoto, “Illustrative Photofabrication”, Sogo-denshi Publication(1985). At first, a sheet-type negative photoresist having a thicknessof 25 μm was applied onto the glass substrate, exposed through aphotomask designed for making the flow path, and the negativephotoresist was developed. Uncured silicone rubber was cast using thenegative-photoresist substrate as a template, and then was cured toproduce a silicon rubber surface having the concave surface with aheight of 25 μm in the region where the electrode was placed.

The electrode substrate and the silicone-rubber flow path were adheredwith a two-fluid-type curing silicone rubber such that the concavesurface of the silicone rubber was faced to the region where theelectrode on the electrode substrate was placed. A syringe for injectinga solution was placed upstream of the flow path, and an apparatusallowing a solution in which the molecules were dissolved to flow on theelectrode was added to the electrode substrate.

Example 4 Measurement of Collecting Rate with Respect to Bovine-SerumAlbumin (BSA) Protein

An electrode formed with a communication groove having the depth of 2 μmor 4 μm was prepared as in Example 3, a flow path was prepared as inReference Example 2, a dielectrophoretic chromatography device of thepresent invention was prepared, and the collecting rate of the devicewas measured in the following manner. For the purpose of comparison,with respect to the dielectrophoretic chromatography device preparedsimilarly except that a communication groove is not formed, thecollecting rate was also measured.

(Sample)

As a sample, a solution containing FITC labeled BSA (molecular weight:approximately 65 kD) (60 μg/ml)was used.

(Operation)

For preventing adsorption of protein molecules to the electrodesubstrate or flow path, a block A (manufactured by Snow Brand MilkProducts CO., Ltd.) was used to block the surface of the flow path,after which FITC labeled BSA was applied to the dielectrophoreticchromatography device.

The average speed of the sample used was 556 μm/sec., and the electricfield was applied for 30 to 120 seconds from a start of measurement. Thecollecting rate was measured with respect to the electric field strengthapplied at that time of 2.14 Mv/m, 2.5 Mv/m, and 2.86 Mv/m.

The measurement of the collecting rate was obtained by the followingEquation.

Collecting rate (%)=[(I ₀ −I _(min))×100]/(I ₀ −I _(back))

Wherein I₀ represents the fixed value of the fluorescent strength beforeapplication of electric field, I_(min) represents the minimum value ofthe fluorescent strength during application of electric field, andI_(back) represents the background.

(Results)

FIG. 20 shows the results. In FIG. 20, there is shown the resultsobtained by the use of the dielectrophoretic chromatography device of-Δ- (depth 4 μm), -□- (depth 2 μm), and -⋄- (depth 0 μm).

As is clear from the results shown in FIG. 20, the deeper the depth ofgroove, the collecting rate (%) enhances. In 2.86 Mv/m, the collectingrate of the apparatus of the present invention having the communicationgroove of 4 μm is 40% as compared with the collecting rate 28% of theconventional apparatus having no communication groove, and thecollecting rate was enhanced by about 43%, in other words, thecollecting ability of the substances intended is remarkably enhanced bythe use of the apparatus according to the present invention.

Example 5 Measurement of Collecting Rate to 500 bpDNA

500 bpDNA labeled by intercalator fluorescent dye YOYO-1 (MolecularProbe Ltd.) was used as a sample. The collecting rate (%) was measuredby the dielectrophophoretic chromatography device of the depth ofgroove, 0 μm, 2 μm and 4 μm. FIG. 21 shows the results.

In FIG. 21, there is shown the results obtained by the use of thedielectrophororetic chromatography device having the communicationgroove of -Δ- (depth 4 μm), -□- (depth 2 μm), and -⋄- (depth 0 μm).

As is clear from the results shown in FIG. 21, Also in this case, in theelectric field strength of 1.5 Mv/m or more, the collecting rate of theapparatus of the present invention having the communication groove ofdepth 4 μm was enhanced by about 20% as compared with the conventionalapparatus having no communication groove.

ADVANTAGEOUS EFFECT OF THE INVENTION

According to the invention 1, since the substances to be measured can beconcentrated (gathered) in the hollow space of the electrode or in theupper and lower directions thereof, the electrode is not present underthe substances to be measured, and therefore, where the fluorescentstrength is detected, the reflection of the excitation light by theelectrode under the measured substances is avoided. As a result, thebackground is reduced, the S/N ratio is enhanced, and the measurementsensitivity is enhanced. Further, the measurement can be made from thelower surface of the electrode. Further, according to the presentinvention, since the measurement can be made from the lower surface, itis possible to measure the substances to be measured by the absorbancethat has been impossible in prior art.

When the measurement is made from the lower surface of the electrode,since the parts other than the region where the substances to bemeasured are concentrated are covered with the electrode, whereby insaid parts the excitation light irradiated from the upper surface doesnot reach the lower surface, the background is reduced, the S/N ratio isenhanced and the measurement sensitivity is enhanced (slit effect). Thisis an extremely great advantage.

According to the invention 2, the provision of lower level places thanelectrode level between or among electrodes which has not at all beendone in prior art leads to the remarkable enhancement of the collectingability (rate) which has a very important role for separation ofsubstances by the dielectrophoresis, which is an enormous effect. Thisis therefore an extremely epoch-making invention.

1-5. (canceled)
 6. A method for separating substances in a liquid,comprising the steps of: providing two or more dielectrophoreticelectrodes, wherein the dielectrophoretic electrodes are at an electrodelevel on a substrate and the places among the electrodes on thesubstrate are at lower levels than the electrode level, so as togenerate a non-uniform electric field, subjecting the liquid containingthe substances to be separated in the non-uniform electric fieldgenerated by applying voltage to the dielectrophoretic electrodes, andseparating the substances by utilizing difference in thedielectrophoretic forces exerting on said substances.
 7. The method ofclaim 6, wherein: the step of subjecting the liquid containingsubstances to be separated in the non-uniform electric field comprisescausing the liquid to flow into the non-uniform electric field generatedby the dielectrophoretic electrodes, and separation is carried out by aninteraction of the dielectrophoretic force exerting on said substanceand fluid drag.
 8. The method according to claim 7 wherein saiddielectrophoretic electrodes are held by a convex construction on saidsubstrate to make the places among said dielectrophoretic electrodes atlower levels than said electrode level.
 9. The method according to claim6, wherein the places among said electrodes at lower levels than saidelectrode level are formed by excavating the substrate with physical orchemical means.
 10. The method according to claim 9, wherein the placesamong said electrodes at lower levels than said electrode level areformed by excavating the substrate with a chemical means, and whereinsaid chemical means is an etching using an etching liquid for thesubstrate.